Regulation of an alternative sigma factor σI by a partner switching mechanism with an anti-sigma factor PrsI and an anti-anti-sigma factor ArsI in Streptomyces coelicolor A3(2)

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376-78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211-213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.     

Characterization of Temperate Actinophage φC31 Isolated from Streptomyces coelicolor A3(2)

Actinophage phiC31 isolated from Streptomyces coelicolor A3(2), the only strain among actinomycetes for which a genetic map had been constructed, appears to be a typical temperate phage. After phiC31 infection, true lysogenic cultures arose which liberated phage and were immune to infection with homologous phage after repeated single-colony isolations and treatment with phage-specific antiserum. Clear-plaque (c) mutants were derived from phiC31 phage which failed to lysogenize sensitive cultures. Actinophage phiC31 has a temperature-sensitive stage of reproduction. A phage which reproduces with the same effectiveness at high (37 C) and low (28 C) temperatures has also been obtained. Heat-inducible (ct) mutants were isolated from this phage which were able to lysogenize sensitive cultures at 28 C but failed to do so at 37 C. Properties of ct mutants suggest that ct mutations involve a gene controlling maintenance of the lysogenic state in actinomycetes and synthesizing repressor, which may become heat-sensitive as a result of mutation.     

Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.     

What type of glutamine synthetase is important forStreptomyces coelicolor A3(2) under nitrogen-limited growth conditions?

Summary Glutamine synthetase I activity ofStreptomyces coelicolor was strongly repressed by ammonia and was induced 56.8 fold in a nitrogen-free medium. Glutamine synthetase II activity was not induced even by a long-term nitrogen starvation. Therefore, glutamine synthetase I is the only active enzyme ofStreptomyces coelicolor.     

Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.     

Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.     

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.     

High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.     

CK2α is essential for embryonic morphogenesis

CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.     

Krüppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation

Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p<0.05, studens t-test). As demonstrated for some other non-glial cancer entities, KLF8-knockdown by shRNA in U87-MG cells confirmed its functional relevance, leading to an almost complete loss of tumor cell proliferation. Selective blocking of KLF8 might represent a novel anti-proliferative treatment strategy for malignant gliomas. Yet, its simultaneous expression in non-proliferating tissues could hamper this approach.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

Inactivation of glycogen synthase kinase-3β is required for osteoclast differentiation

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.     

Ets-1 is essential for connective tissue growth factor (CTGF/CCN2) induction by TGF-β1 in osteoblasts

Background

Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts.

Results

We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3.

Conclusions

This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.